Cytosol fractions were prepared from fetal rat or embryonic chick calvaria and analyzed for binding of vitamin D3 metabolites on sucrose density gradients. Both cytosol fractions contain a 3.5S macromolecule which specifically binds 1,25-dihydroxy-[3H]vitamin D3 and in addition, a 5 to 6S macromolecule which binds 25-hydroxy-[3H]vitamin D3. In rat calvaria cytosol, 1,25-dihydroxy-[3H]vitamin D3 also binds to the 5 to 6S macromolecule but appears to have greater affinity for the 3.5S component.
A competitive binding assay for 1,25-dihydroxyvitamin D [1,25-(OH2D] in plasma has been developed in which intestinal cytosol preparations from rachitic chicks are used as the binding protein. A new method of extraction and two new chromatographic procedures are used for this assay. The method is sensitive to as little as 10 picograms of 1,25-(OH)2D, and triplicate assays can be done on 5 milliliters of plasma. This assay shows that in the plasma of normal adult subjects there is a 1,25-(OH)2D concentration of 29 +/- 2 picograms per milliliter, while none can be detected in the plasma of nephrectomized subjects and end-stage renal failure patients.
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